Project Summary Ethanol disrupts hepatic function with the eventual appearance of alcoholic liver disease. Kupffer cells, the resident macrophages in the liver, are critical to the onset of ethanol-induced liver injury. Activation of macrophages by endotoxin/ lipopolysaccharide (LPS), a component of the cell wall of gram-negative bacteria, via the toll-like receptor 4 (TLR-4) leads to the production of a variety of inflammatory mediators, such as tumor necrosis factor-[unreadable] (TNF-[unreadable]) and reactive oxygen species. During chronic ethanol exposure, abnormal production of TNF-[unreadable] is a critical component in the development of inflammation in the liver. Increased TNF-[unreadable] production during ethanol exposure is due to both an increased exposure to LPS, as well as a sensitization Kupffer cells to activation by LPS. This ethanol-induced sensitization results from changes in the regulation of TLR-4 signal transduction, culminating in the dysregulation of transcriptional and post-transcriptional control of TNF-[unreadable] expression. The long- term goals of this research project are to understand the molecular mechanisms by which chronic ethanol sensitizes Kupffer cells to TLR-4 mediated signaling. In the past granting period, we have characterized the molecular mechanisms for ethanol-induced sensitization of LPS-stimulated TNF-[unreadable] expression, focusing on regulation of mRNA stability. More recently, we have investigated the interaction between adiponectin, a potent anti-inflammatory adipokine, and Kupffer cells during chronic ethanol exposure. Treatment of Kupffer cells with adiponectin normalizes the increased sensitivity to LPS in Kupffer cells isolated from ethanol-fed rats. Importantly, Kupffer cells from ethanol-fed rats exhibited increased sensitivity to the anti-inflammatory effects of adiponectin. The major objective of this proposal is to investigate the molecular mechanisms for the anti-inflammatory effects of adiponectin. Utilizing primary cultures of Kupffer cells from ethanol- and pair-fed rats, the Specific Aims of this proposal will test the following hypotheses: 1) The anti-inflammatory effects of adiponectin on Kupffer cells are due to increased expression of the anti-inflammatory cytokine, IL-10. Further, we hypothesize that adiponectin- stimulated IL-10 production will be higher in Kupffer cells from ethanol-fed rats compared to pair-fed controls. 2) Desensitization of LPS-stimulated signal transduction by adiponectin occurs at proximal TLR-4 signaling events, leading to a normalization of LPS-stimulated NADPH oxidase activity in Kupffer cells from ethanol-fed rats. 3) Adiponectin suppresses/normalizes LPS-stimulated TNF-[unreadable] expression in Kupffer cells via transcriptional and post- transcriptional regulatory mechanisms, involving epigenetic regulation and control of mRNA stability, respectively. Understanding the molecular mechanisms for the anti-inflammatory effects of adiponectin in Kupffer cells after chronic ethanol exposure will enable the design of therapeutic strategies aimed at slowing or reversing the progression of alcoholic liver disease.